This invention relates to novel electrophoretic media. The media preferably comprise polymer gels which exhibit greater strength, resolution and recoverability of separated products such as DNA than commercially available gels. The media can also be otherwise formulated, such as in bead form and as a surface coating.
During the last decade, considerable advances have been made in molecular biology revolving around the ability to manipulate peptides, DNA and RNA. These advances have fueled the emergence of the biotechnology industry, with extensive research and development geared to the production of biopharmaceuticals, genetically engineered vaccines, immunochemicals, organisms, plants and novel diagnostics. Electrophoresis, a technique in which complex biological substances such as proteins, peptides, DNA and RNA are separated according to size and/or charge, is a powerful separation method widely used within every life science discipline. The procedure is used for the resolution and isolation of complex biological substances such as proteins, peptides, DNA and RNA, and is thus a technique upon which the emerging biotechnology industry is greatly dependent. The needs of the industry have placed new and increased demands on electrophoretic technology, there being a considerable need for electrophoretic media which can provide improved resolution, handleability, and recovery and a range of matrix pore sizes which can be used in newly discovered applications. Most analytical electrophoresis methods are based on zone electrophoresis in which a thin zone of a sample is applied to the electrophoretic medium. When the components of the sample are to be separated according to their charge, an electric potential is applied to the electrophoretic medium for a certain period of time, so that charged components of the sample move in various distances depending on their chemical natures. When the components of the sample are to be separated according to their size, the electrophoretic medium contains a denaturing agent so that components of the sample move in various distances depending on their molecular weights. The migration of the sample components results in the formation of fractional zones which can then be examined and studied by application of standard electrophoretic practices such as fixing, staining, and washing to remove buffers. Typically, the electrophoretic medium is a thin gel slab supported by a suitable material, commonly glass or plastic.
Various hydrophilic colloids, such as starch, cellulose acetate and agarose have been used in the forming of electrophoretic gel slabs, but polyacrylamide is generally favored. Polyacrylamide is used as a cast material composed of varying amounts of acrylamide and bis-acrylamide. N,N.sup.1 -bis-acrylylcystamine, N,N.sup.1 -dihydroxy ethylene bis-acrylamide, and N,N.sup.1 -diallyltartardiamide have also been used. These materials are conventionally proportioned to prepare, on polymerization, a network of polymeric fibers for sieving or anti-convection. Viscosity of the gel is adjusted by varying the amounts of acrylamide and bis-acrylamide. Frequently used catalyst and initiator are TEMED (tetraethylaminediamine) and ammonium persulfate or riboflavin/light.
Methods known in the art for utilizing polyacrylamide gels for determination of nucleotide sequences involve the preparation of the gels in given thicknesses, such as between glass plates to a thickness of approximately 0.3 mm. In some applications the gel may be polymerized onto a support film. DNA samples labeled such as with .sup.32 P, .sup.35 S or fluorescent dyes are placed onto sample slots and electrophoresed. After electrophoresis (1-24 hours) the gel is removed from the glass plates and autoradiography performed. In automated systems, fluorescent labeled nucleotides are monitored during the separation. Autoradiography requires 10 to 20 hours after which time films are studied to determine nucleotide sequence. The preparation of gels for autoradiography of .sup.35 S nucleotides requires immersion in 10% acetic acid to remove urea and handling of the gels with caution due to extreme fragility.
When proteins are being separated by electrophoretic methods based on their size, sodium dodecyl sulfate (SDS) is generally added to the polyacrylamide gel alone, or in conjunction with other denaturants, to unfold the protein and provide a net negative charge. Molecular sizes can be estimated from mobilities as compared to known standards. When separations are being made according to charge, the polyacrylamide gels are generally used in combination with acidic, basic or neutral buffer systems in the absence of denaturing agents. Electrodes are positioned according to the predicted net charge of the sample at the pH used.
Despite the widespread use of polyacrylamide gels to separate complex proteins, double or single stranded DNA, synthetic oligonucleotides and the like as well as for DNA sequencing, a number of disadvantages are associated with polyacrylamide. Among them are neurotoxicity, short shelf life, cumbersome preparation, and gel fragility. Neurotoxicity and instability have only recently been addressed in the development of adequate precast polyacrylamide gels. Gel fragility is considered a major difficulty in DNA sequencing where ultrathin gels are required for optimum resolution on autoradiography of radiolabeled nucleotides. These disadvantages are also found in other applications of electrophoresis such as separation of proteins.
Recognizing the shortcomings of polyacrylamide gels, many have attempted to improve the gels. U.S. Pat. No. 4,657,656 to Ogawa discloses an improved medium for electrophoresis comprising a polyacrylamide gel formed by crosslinking polymerization of an acrylamide compound and a crosslinking agent and further containing a water soluble polymer having a molecular weight in the range of 10,000 to 1,000,000, such as polyvinyl alcohol or polyacrylamide. Incorporation of the water soluble polymer such as solid polyacrylamide is said to reduce the brittleness of the polyacrylamide gel. Crosslinking agents disclosed as being suitable are N,N'-methylenebisacrylamide, N,N'-propylenebisacrylamide, diacrylamide dimethylether, 1,2-diacrylamide ethyleneglycol, ethyleneureabisacrylamide, ethylene diacrylate,N,N'-diallyltartardiamide and N,N'-bisacrylylcystamine.
U.S. Pat. No. 4,695,354 to Sugihara et al. discloses that conventional thin polyacrylamide gels are unsuitable because, when used to resolve nucleic acid fragments, they give distorted patterns. Sugihara et al. disclose that the resolution of the gels can be improved by incorporating into the gels less than 1 wt/v% of glycerol.
The fragility and brittleness of conventional polyacrylamide gel membranes can lead to problems when it is desired to dry the membranes for enhanced resolution. As disclosed in U.S. Pat. No. 4,699,705 to Ogawa et al., in the drying process, the adhesion between the glass plate and the membrane is negligible, the membrane is easily broken. To alleviate these problems, Ogawa et al. disclose that the adhesion between the membrane and its support can be enhanced by utilizing as the support a polymer sheet which has been subjected to glow discharge treatment. The patent also suggests the incorporation in the gel medium of at least one carbamoyl group-containing compound, such as urea or formamide, as modifier. Other methods disclosed for improving the adhesion between a polyacrylamide gel membrane and its support involve the use of special adhesives as disclosed in U.S. Pat. Nos. 4,548,869, 4,548,870, 4,579,783 and U.S. Pat. No. 4,600,641 to Ogawa et al. and in U.S. Pat. No. 4,415,428 to Nochumson et al.
U.S. Pat. No. 4,582,868 to Ogawa et al. notes that the polymerization reaction for the preparation of polyacrylamide can be inhibited by the presence of oxygen. It discloses a novel medium for electrophoresis in the form of an aqueous gel which can be prepared in the presence of oxygen. The novel medium is an acrylamide copolymer having a specifically selected repeating unit.
U.S Pat. No. 4,189,370 to Boschetti discloses gel polymers prepared by radical polymerization of N-methylol-acrylamide and a bifunctional allylic or acrylic compound causing cross-linking to yield a tridimensional configuration polymer. Examples of cross-linking agents disclosed in the patent are N,N'-methylene bisacrylamide, diallyltartramide and ethylenediacrylate.
Despite the great amount of effort which has gone into improving conventional polyacrylamide gels, there is still a need for new gels which overcome the problems associated with acrylamide gels such as brittleness, neurotoxicity, cumbersome preparation and short shelf life. There is also a need for new gels which have greater resolution power and recoverability of separated DNA and protein materials to meet the demands of the emerging biotechnology industry.